DESCRIPTION: This proposal requests continued support for the PI's long-standing investigation into the mechanical properties of blood cells, and how these properties are altered by abnormalities in the structural components of the membrane. The current proposal focuses on the role of the association between the bilayer and the underlying membrane skeleton. This is a particularly important process in red cells, which must maintain their integrity while undergoing large cyclic deformations. The proposed approach involves direct micromechanical experiments on individual cells with specific abnormalities, making direct measurements of the forces needed to separate the membrane bilayer from the membrane skeleton. These results are analyzed in the framework of specific models, which enable quantitative predictions of how structural changes affect the separation process. The major linkages to be examined are those involving protein 3 and its connections with ankyrin, protein 4.1, and protein 4.2. Four specific aims are proposed. In AIM1, he will measure the effects of selected chemical modifications on the strength of the bilayer-skeletal association in red cells. Cells with naturally occurring deficiencies, or mutations introduced by gene knockout and Transgenic strategies will be used in these experiments. The effects of oxidative damage on the separation process will also be investigated. The PI will use fluorescent markers to measure the lateral distribution of membrane components during and after tether formation, a technique in which the force required to extrude a thin finger of membrane from a cell's surface, to obtain a detailed assessment of the molecular events that accompany this process. In AIM2, the PI seeks to refine quantitative models of the mechanics of bilayer detachment, in order to provide a rational basis for experimental design and interpretation. He hypotheses that the bilayer skeletal detachment force depends critically on the lateral spacing of molecular bonds between the skeleton and the bilayer, and the rigidity of the skeleton. In AIM3, they extend their studies to consider how the attachment of membrane proteins to a skeleton or substrate affects their distribution between the cell body (with a skeleton) and the tether (which lacks a skeleton). The role of these forces in mediating the lateral distribution of integral membrane proteins will also be examined. Finally, in AIM4, they will apply these techniques to understand the adhesive interactions of lymphocytes, comparing the contributions of skeletal attachment to the adhesion of receptors that mediate rolling (selectin) vs. those that stabilize fixed attachments (LFA-1). Collectively, these studies will provide important new understandings of the forces that shape and stabilize the membrane, and of the role of the membrane skeleton in mediating cell adhesion receptors. These understandings are important to fully comprehend the molecular basis of various hemolytic and inflammatory disorders, and compliment the many biochemical and structural evaluations of this structure.